Numerous transcription factors allow hematopoietic cells to respond to lineage- and stage-specific cytokines and/or to act as their effectors. The transcription factors PU.1 and c-Myb are essential for hematopoiesis, most likely acting at distinct stages of differentiation, but sharing a common set of target genes. To determine whether PU.1 and c-Myb are functionally interrelated, murine bone marrow (BM) cells and 32Dcl3 murine myeloid precursor cells were infected with a retrovirus carrying a PU.1 cDNA and assessed for myeloid colony formation and for granulocytic differentiation, respectively. Compared with noninfected normal BM cells or to cells infected with an empty virus, hematopoietic precursor cells expressing PU.1 formed an increased number of interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF)-stimulated colonies. Moreover, granulocytic differentiation of 32Dcl3 cells constitutively expressing PU.1 was accelerated, as indicated by morphology and by expression of differentiation markers. Downregulation of c-Myb protein levels by expression of an antisense c-myb construct was also associated with a faster kinetics of 32Dcl3 granulocytic differentiation. Sequence analysis of the 5' flanking region of the c-myb gene revealed a consensus PU box at position +16 to +21 able to specifically interact in electrophoretic mobility shift assays with either bacterially synthesized PU.1 protein or whole cell extracts from differentiated 32Dcl3 cells. Transient expression of PU.1 in cotransfection assays in different cell lines resulted in inhibition of chloramphenicol acetyl transferase activity driven by different segments of the c-myb promoter. Moreover, such an effect was dependent on an intact PU box. Thus, the ability of PU.1 to potentiate terminal myeloid differentiation appears to involve downregulation of c-myb expression, an essential step during differentiation of hematopoietic precursor cells.